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  • CHRONIC ITCH

    Gate control of mechanical itch by a subpopulation of spinal cord interneurons Steeve Bourane,1* Bo Duan,2* Stephanie C. Koch,1 Antoine Dalet,1 Olivier Britz,1

    Lidia Garcia-Campmany,1 Euiseok Kim,3 Longzhen Cheng,2,4 Anirvan Ghosh,3

    Qiufu Ma,2† Martyn Goulding1†

    Light mechanical stimulation of hairy skin can induce a form of itch known as mechanical itch. This itch sensation is normally suppressed by inputs from mechanoreceptors; however, in many forms of chronic itch, including alloknesis, this gating mechanism is lost. Here we demonstrate that a population of spinal inhibitory interneurons that are defined by the expression of neuropeptide Y::Cre (NPY::Cre) act to gate mechanical itch. Mice in which dorsal NPY::Cre-derived neurons are selectively ablated or silenced develop mechanical itch without an increase in sensitivity to chemical itch or pain. This chronic itch state is histamine-independent and is transmitted independently of neurons that express the gastrin-releasing peptide receptor. Thus, our studies reveal a dedicated spinal cord inhibitory pathway that gates the transmission of mechanical itch.

    T he sensation of itch elicits stereotypical scratching behaviors that are an impor- tant protective response to cutaneous irri- tants and parasites. Animals appear to have evolved two forms of itch: (i) chemical itch,

    which is activated by chemical mediators such as histamines and proteases (1–6) and can be effec- tively gated by noxious painful stimuli (7), and (ii) mechanical itch, which is evoked by light tac-

    tile stimuli, such as when insects or parasites come in contact with the skin. In humans, this latter pathway can be activated by vibrating the fine vellous hair (8). Itching is also frequently evoked by light mechanical stimuli in patients suffering from chronic itch (9, 10). Although progress has beenmade toward iden-

    tifying the spinal inhibitory neurons that gate chemical itch (11, 12), little is known about the

    spinal pathways gating mechanical itch. The dor- sal horn of the spinal cord contains multiple in- hibitory interneuron (IN) populations including cells that express neuropeptide Y (NPY) (13, 14). These cells are distinct from those that express dynorphin, galanin, neuronal nitric oxide syn- thase (nNOS), and parvalbumin (15, 16). When NPY::Cre transgenic mice [Gene Expression Nervous System Atlas (GENSAT), RH26 cell line (see supplementary materials and methods)] were crossed with R26LSL-tdTomato (ai14) reporter mice to trace the provenance of the dorsal horn INs expressing NPY, NPY::Cre-derived INs were localized in laminae III and IV (70.4 ± 0.3%) and, to a lesser extent, in laminae I and II (29.6 ± 0.3%) (Fig. 1, A and B). The number of NPY+/ tdTomato+ INs decreases postnatally, with only 35% of the tdTomato cells expressing NPY at postnatal day 30 (P30) (Fig. 1C). NPY::Cre thus captures two populations of NPY-expressing neu- rons: one that transiently expresses NPY during late embryonic and early neonatal development

    550 30 OCTOBER 2015 • VOL 350 ISSUE 6260 sciencemag.org SCIENCE

    NPY::Cre; R26 LSL-tdTomato CGRP IB4 PKCγ

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    Fig. 1. NPY::Cre delineates a population of inhibitory neurons in the dor- sal spinal cord. (A and B) Sections through the lumbar dorsal spinal cord of a P30 NPY::Cre; R26LSL-tdTomato mouse stained with calcitonin gene-related peptide (CGRP) and IB4 (A) and protein kinase Cg (PKCg) (B). tdTomato+ fluorescence was visualized without staining. Roman numerals denote laminae. (C) A section through the lumbar dorsal spinal cord of a P30 NPY::Cre; R26LSL-tdTomato

    mouse, comparing expression of tdTomato reporter (red) and NPY (green, in situ). Arrows indicate double-labeled cells. (D) A section through the lumbar dorsal horn of a P30 NPY::Cre; R26LSL-tdTomato, showing coexpression of tdTomato with the inhibitory markers Gad1 and GlyT2. (E) Quantification of coexpression of NPY-tdTomato+ with Gad1-GFP, GlyT2-GFP, and Gad1+/GlyT2

    in situ hybridization. (F) Firing properties of NPY::Cre INs.The majority of NPY- tdTomato+ INs (34 of 42 cells) display a tonic firing pattern upon current injection. IB, intermittent bursting; TwG, tonic firing with gap. (G to L) In situ analysis and quantification of NPY [(G) to (I)] and Gad1/GlyT2 [(J) to (L)] expression in the lumbar dorsal spinal cord of P60 control and NPY::Cre IN– ablatedmice. NPY+ cell numbers were reduced by 59.0 and 69.5% in laminae I and II and laminae III and IV, respectively [(I) ***P < 0.001]. Gad1+ and GlyT2+

    cell numbers were reduced by 34.7% in laminae I and II [(L) *P < 0.05] and 34.9% in laminae III and IV [(L) **P < 0.01]. P values were calculated using the Student’s unpaired t test. Scale bars: 50 mm [(C), (D), (H), and (K)]; 200 mm [(A) and (B)]. Error bars indicate SEM [(I) and (L)].

    1Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. 2Dana-Farber Cancer Institute and Department of Neurobiology, Harvard Medical School, 1 Jimmy Fund Way, Boston, MA 02115, USA. 3Neurobiology Section, Division of Biological Sciences, University of California, San Diego, CA 92093, USA. 4Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China. *These authors contributed equally to this work. †Corresponding author. E-mail: goulding@salk.edu (M.G.); qiufu_ma@dfci.harvard.edu (Q.M.)

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  • and another that shows persistent expression into adulthood. More than 98% of the NPY::Cre-tdTomato+

    cells expressedglutamic acid decarboxylase 1 (Gad1) and/or the glycine transporter 2 (GlyT2+) (Fig. 1, D and E). After current injection, the majority of cells (34 of 42) displayed a tonic firing pattern (Fig. 1F) that is characteristic of many dorsal horn inhibitory INs (17). These NPY::Cre INs make up 31 and 45% of the inhibitory Gad1+ or GlyT2+ INs in laminae I and II, and in laminae III and IV, respectively. Very few of the NPY:: Cre INs cells expressed nNOS (4.9 ± 0.6%), galanin (5.0 ± 1.1%), dynorphin (8.2 ± 1.6%), or parvalbumin (5.9 ± 1.7%), indicating that they constitute a distinct population of dorsal horn inhibitory INs. An intersectional genetic strategy that restricts

    diphtheria toxin receptor (DTR) expression to NPY::Cre-derived INs in the dorsal spinal cord andmedulla (18) was then used to determine the contribution the NPY::Cre INs make to gating cutaneous sensory stimuli. Injecting NPY::Cre; Lbx1FlpO; Tauds-DTR mice with diphtheria toxin (DTX) markedly reduced the number of NPY:: Cre-tdTomato INs in the dorsal spinal cord (fig. S1, A to C). This cell loss was restricted to inhib-

    itory INs that express NPY, Gad1 and/or GlyT2 (Fig. 1, G to L, and fig. S1, G to I), and Pax2 (fig. S1, D to F). Neighboring dorsal inhibitory IN sub- types expressing nNOS, dynorphin, and parv- albumin (fig. S1, J to R) were spared, as were dorsal excitatory IN subtypes (fig. S2, A to L). There was no noticeable change in the central projections of sensory afferent nerve fibers or in the distribution of NPY::Cre-derived neurons in other regions of the central nervous system (fig. S3, A and B). Twoweeks after injectionwithDTX, the NPY::

    Cre IN–ablated mice began to display spontane- ous scratching, followed by the appearance of skin lesions (Fig. 2, A and B). This scratching was not related to chemical itch, as injection of chemical pruritogens (compound 48/80 and chlo- roquine) into the nape region of NPY::Cre IN– ablated mice before the onset of spontaneous scratching revealed no difference in the level or intensity of scratching (Fig. 2, C and D). Follow- ing a modified protocol for analyzing alloknesis in mice in which von Frey hairs were used to deliver graded mechanical forces to the nape of the neck (19) (Fig. 2E), we observed a significant increase in evoked hindlimb scratching in NPY:: Cre IN–ablatedmice with low-force (0.02 to 0.4 g)

    von Frey hairs as compared with control mice (Fig. 2F). In contrast, high-threshold mechanical stimuli (0.6 to 1 g) did not induce pronounced scratching. Additional behavioral tests revealed nomarked differences between control andNPY:: Cre IN–ablated mice with regard to their respon- siveness to noxiousmechanical and thermal stimuli (fig. S4A). Acute chemical pain sensitivity was also normal: Injection of capsaicin into the cheek of control and NPY::Cre IN–ablated mice pro- duced similar levels of pain-indicating wiping, with little or no itch-indicating scratching (20) (fig. S4B). To rule out the